Ligand-receptor assays utilize the exquisite specificity and sensitivity of bioreagents to identify and quantify minute amounts of a wide range of substances also referred to as analytes. Competitive ligand-receptor assays are one variant of ligand-receptor assays in general. In competitive ligand-receptor assays, analyte substances in the sample compete with a signal-producing substance for a limited number of binding sites on the counterpart of the ligand-receptor pair. After the binding has taken place, the amount of signal-producing substance bound to the counterpart is detected by several different means. The signal intensity of competitive ligand-receptor assay is in an inverse relationship with the concentration of analyte present; thus, a sample with no analyte will give a maximum signal intensity, and a sample with a range of analyte concentration will produce less than a maximum signal. Competitive ligand-receptor assays can be adapted to many different types of signal-producing systems: enzymes, radioisotopes, fluorescent compounds, colored latex particles, and the like. The signal-producing means, however, does not alter the inverse relationship between signal intensity and analyte concentration (FIG. 1). Conventional competitive ligand-receptor assays to date have several disadvantages. First, such an assay has its maximum sensitivity in a narrow analyte concentration range, around the inflection point of the signal intensity versus analyte concentration curve (FIG. 1). This sensitivity parameter, defined as the change in signal intensity per unit change in analyte concentration (.DELTA.signal/A.DELTA.[analyte]), decreases significantly as the analyte concentration is farther from the inflection point of the curve. The maximum sensitivity of the assay is at the concentration which gives 50% of the maximum signal intensity. Furthermore, changing the maximum sensitivity range of a competitive assay has to date proven very difficult. Changing such a range requires careful experimentation as to the new amounts of reagents supplied for the assay, as well as a careful recalibration with an external source.
Yet another major disadvantage of competitive ligand-receptor assays is the absolute need for calibration. This disadvantage manifests itself in so-called semi-quantitative assays, where a "yes" or "no" is indicated by the assay based on the presence or absence of a predetermined concentration of analyte. Such semi-quantitative competitive ligand-receptor assays are difficult to perform without external calibration, thus limiting their usefulness in a variety of important market segments. Due to the inverse relationship between signal intensity and analyte concentration, all but the most concentrated samples will give a signal in the assay, and therefore a standard curve (or at least one control point with a known standard) must be run in parallel with the sample assay to interpret accurately any reading of the sample assay. For example, an optical density reading of 0.5 in a competitive immunoassay using enzymes as a signal producing system is meaningless. However, if the user runs a known standard of, for example, 10 micrograms per milliliter of analyte and obtains a reading of 1.0, then the sample with the reading of 0.5 can be said to be more concentrated than the 10 microgram per milliliter sample. The need for standardization has severely limited the practical usefulness of current competitive ligand-receptor assays by requiring several runs of the assay to determine one sample concentration. One of the major disadvantages of the requirement for outside calibration is the concomitant reduction of precision and accuracy of each assay due to inter-assay variability in the calibration process. Furthermore, while there are commercially-available immunochromatographic test strip versions of the ligand-receptor competitive assay available that do not require external calibration, these assays are designed to give a positive indication for the analyte at the least sensitive portion of the analyte concentration versus signal intensity curve. Thus, these immunochromatographic test strips are to be interpreted as positive for analyte in the sample when no signal is seen at the test line. This signal intensity would correspond to the highest concentration range in the dose response curve set forth in FIG. 1. As one skilled in the art would recognize, the precision of the determination of analyte concentration is compromised in such an assay.
Thus, there exists a need for a competitive assay which retains the advantages of ligand-receptor tests (i.e., both specificity and sensitivity) for minute amounts of analyte, while improving the ratio of .DELTA.signal/.DELTA.[analyte] in a wide range of analyte concentrations. Furthermore, a need exists for such an assay where the operator does not have to use an external calibration. As an extension for this need, there exists a need for competitive ligand-receptor assays wherein the maximum of sensitivity can be easily shifted and a semi-quantitative assay with great precision is available without the need for external calibration. The present invention provides these advantages and more.